Project Summary Approximately 20% of colorectal cancers (CRCs), representing 25,000 patients in the U.S. each year, harbor activating driver mutations in the PIK3CA gene, which encodes the catalytic alpha subunit of phosphatidylinositol-3-kinase (PI3K). These patients have particularly poor prognoses when treated with standard-of-care chemotherapies and also fail to respond to single agent inhibitors of the PI3K pathway, a problem that underscores the compelling need for more effective therapeutic strategies for PIK3CA mutant CRC. Recently, by combining phosphoproteomic data from human CRC patients with combinatorial drug screening in CRC cell lines, we discovered that inhibitors of the extracellular signal-regulated kinase (ERK) pathway selectively sensitize PIK3CA mutant CRCs to inhibitors of the tyrosine kinase SRC without regard to the mutational status of other commonly mutated genes, including KRAS and BRAF. Through mechanistic studies, it was revealed that the selectivity of this combination for PIK3CA mutant CRCs owes to (1) SRC's selective activation only in PIK3CA mutant cells, where its pharmacological inhibition cooperates with ERK pathway inhibition to trigger apoptosis through the induction of the pro-apoptotic protein BIM, and (2) the fact that PIK3CA mutant CRCs are ?addicted? to chronic BIM suppression driven by constitutive PI3K activation, and are thus vastly more sensitive to BIM induction than their PIK3CA wild-type counterparts. Acquired resistance to combinations of SRC and ERK pathway inhibitors converges on the activation of the anti- apoptotic protein BCL-XL, which directly antagonizes BIM. Upfront inhibition of BCL-XL in combination with SRC and ERK pathway inhibition leads to penetrant cell killing while blocking the emergence of acquired resistance. In this proposal, we build on these findings through a series of integrated studies examining (1) the molecular mechanisms governing SRC activation and its cooperation with the ERK pathway in PIK3CA mutant CRC; (2) the translational potential of combined SRC plus ERK pathway inhibition using studies in patient- derived xenograft (PDX) models alongside correlative clinical studies in human patient tumors; and (3) the ability of BCL-XL inhibition to reverse or block acquired resistance, alongside studies to elucidate the molecular mechanisms governing BCL-XL activation in the resistant state. The work will define an essential survival signaling network in PIK3CA mutant CRC and is expected to lead to the first selective targeted therapeutic strategies for this aggressive disease subtype.